Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.